25-hydroxyvitamin D3 (25-OH D3), or calcidiol/calcifediol, is a pro-hormone for calcitriol that restores bone mineralization in vitamin D-deficient models. 25-OH D3 is the major circulating metabolite of vitamin D3. Vitamin D signaling also regulates Ca2+ homeostasis. 25-OH D3 activates the vitamin D receptor and also regulates transcription of hormones such as parathyroid hormone (PTH). In vivo, 25-OH D3 exhibits anti-inflammatory activity, improving diabetic periodontitis; 25-OH D3 decreases levels of serum glucose, glycosylated hemoglobin, and TNF-α and downregulates expression of toll-like receptor 4 (TLR4), JAK/STAT, and NF-κB. This compound also displays anticancer activity, as it inhibits cell growth in prostate cancer cells.